WO2023192994A2 - Méthode de stimulation de la régénération de cellules ganglionnaires rétiniennes - Google Patents

Méthode de stimulation de la régénération de cellules ganglionnaires rétiniennes Download PDF

Info

Publication number
WO2023192994A2
WO2023192994A2 PCT/US2023/065219 US2023065219W WO2023192994A2 WO 2023192994 A2 WO2023192994 A2 WO 2023192994A2 US 2023065219 W US2023065219 W US 2023065219W WO 2023192994 A2 WO2023192994 A2 WO 2023192994A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
cells
rgc
transcription factor
retinal
Prior art date
Application number
PCT/US2023/065219
Other languages
English (en)
Other versions
WO2023192994A3 (fr
Inventor
Levi J. TODD
Thomas A. Reh
Wesley JENKINS
Marina PAVLOU
Juliette WOHLSCHLEGEL
Original Assignee
University Of Washington
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Washington filed Critical University Of Washington
Publication of WO2023192994A2 publication Critical patent/WO2023192994A2/fr
Publication of WO2023192994A3 publication Critical patent/WO2023192994A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • BACKGROUND Neurodegenerative disorders of the eye result in blindness because the mammalian nervous system lacks a regenerative capacity. In other vertebrates, such as fish and amphibians, the retina is able to replace lost neurons and restore visual function.
  • Müller glia MG
  • the primary glial cell in the vertebrate retina can serve as a source of neurogenic progenitors in regenerative species.
  • MG respond to retinal damage by undergoing an inflammatory response instead of a regenerative one.
  • compositions, nucleic acid molecules and methods for inducing retinal regeneration and reprogramming of Müller glia (MG) into retinal ganglion cells in a subject show that the developmental retinal ganglion cell (RGC) transcription factors Pou4f2 and Islet1 increase the Ascl1-induced neurogenic capacity of MG.
  • RGC retinal ganglion cell
  • Ascl1, Pou4f2 and Islet1 stimulates MG to generate bipolar cells and RGC-like neurons.
  • the transcription factor Onecut1 which is expressed in developing retinal cells, but not in MG, induces MG to generate RCG-like cells.
  • Additional transcription factors that can be used include Irx2, Irx5, Neurod2, Ebf1, and Tcf3.
  • MG- derived RGCs can exhibit action potentials in vivo, and display chromatin profiles similar to developing RGCs.
  • RGC developmental retinal ganglion cell
  • the transcription factor is selected from Onecut1, Pou4f2, and Islet1, and combinations thereof. In some embodiments, the transcription factor is selected from Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof. In some embodiments, the RGC transcription factor is Onecut1. In some embodiments, the RGC transcription factor comprises Pou4f2 and/or Islet1. In some embodiments, the RGC transcription factor comprises Irx2 and/or Neurod2. [0008] In some embodiments, the nucleic acid sequence further comprises a nucleic sequence that encodes a proneural basic helix-loop-helix (bHLH) transcription factor.
  • bHLH proneural basic helix-loop-helix
  • the proneural bHLH transcription factor and the RGC transcription factor are expressed as a fusion protein.
  • Representative examples of proneural bHLH transcription factors include, but are not limited to, Ascl1, Atonal7 (also known as Math5), Atoh1 (also known as Math1), Neurogenin-2, and Neuronal Differentiation 1 (Neurod1).
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding Ascl1 and Atoh1.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding Ascl1 and Atoh7.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding Atoh1 and Atoh7.
  • the nucleic acid molecule further comprises a nucleic acid sequence encoding Ascl1, Atoh1 and Atoh7. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding two, three, four, or all five of Ascl1, Atonal7, Atoh1, Neurogenin-2, and Neurod1. [0009] In some embodiments, the nucleic acid comprises Pou4f2 and/or Islet1 and/or Ascl1. In some embodiments, the nucleic acid comprises Pou4f2, Islet1, and Ascl1. [0010] In some embodiments, the nucleic acid sequence further comprises a promoter sequence in operable linkage with the nucleic acid sequence encoding the RGC transcription factor.
  • the promoter sequence does not naturally occur in operable linkage with the nucleic acid sequence encoding the RGC transcription factor.
  • the promoter sequence is for a gene specifically expressed in glial cells, such as RLBP1 or GLAST.
  • the promoter sequence is a HES1, RLBP1 or GLAST promoter.
  • the promoter sequence will precede an effector gene CRE or tTA that will drive transcription in an inducible manner.
  • the MG-specific promoter sequence is a Rbpl1 promoter sequence or a portion thereof.
  • the nucleic acid sequence comprises an IRES or 2A self- cleaving sites situated between the sequences encoding the transcription factors, for example, in a multicistronic or polycistronic configuration.
  • the nucleic acid sequence will comprise elements that respond to the presence of CRE or tTA, to trigger transcription of genes in an inducible manner. These elements include, but are not limited to, loxP, lox2272 and tetracycline response element (TRE).
  • a composition comprising a nucleic acid molecule described herein.
  • the nucleic acid molecule is an mRNA. Such compositions can be formulated for delivery, optionally in the form of a vector.
  • the vector is a non-viral vector or a viral vector.
  • the viral vector is an adeno- associated viral (AAV) vector or a lentiviral vector.
  • the composition is formulated for administration to the retina. Examples of such administration include, but are not limited to, intravitreal or subretinal injection.
  • the composition further comprises a histone deacetylase (HDAC) inhibitor (HDACi).
  • HDAC histone deacetylase
  • HDACi include, but are not limited to, trichostatin A (TSA), Istodax TM also known as (Pro)/romidepsin, Beleodaq TM , also known as (Pro)/belinostat, Farydak TM , also known as (Pro)/panobinostat, and Zolinza TM , also known as (Pro)/vorinostat.
  • TSA trichostatin A
  • Istodax TM also known as (Pro)/romidepsin
  • Beleodaq TM also known as (Pro)/belinostat
  • Farydak TM also known as (Pro)/panobinostat
  • Zolinza TM also known as (Pro)/vorinostat.
  • Exemplary HDACi peptides include, without limitation, 16cyc- HxA, 16lin-HxA and 16KA (SEQ ID NO: 58-60).
  • Also disclosed herein is a method for inducing retinal regeneration, or a method for stimulating regeneration of a retinal ganglion cell by administering to a cell, e.g., an MG cell, a nucleic acid molecule comprising a nucleic acid sequence encoding a developmental retinal ganglion cell (RGC) transcription factor (TF).
  • a cell e.g., an MG cell
  • a nucleic acid molecule comprising a nucleic acid sequence encoding a developmental retinal ganglion cell (RGC) transcription factor (TF).
  • RGC developmental retinal ganglion cell
  • Exemplary RGC TFs include Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof.
  • the transcription factor is selected from Onecut1, Pou4f2, and Islet1, and combinations thereof.
  • the transcription factor is selected from Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof.
  • the RGC transcription factor is Onecut1.
  • the RGC transcription factor comprises Pou4f2 and/or Islet1.
  • the RGC transcription factor comprises Irx2 and/or Neurod2.
  • Also disclosed herein is a method for inducing retinal regeneration, or a method for stimulating regeneration of retinal ganglion cells, in a subject. These methods comprise administering to a retina of the subject a nucleic acid molecule comprising a nucleic acid sequence encoding a developmental retinal ganglion cell (RGC) transcription factor.
  • RRC developmental retinal ganglion cell
  • Exemplary RGC TFs include Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof.
  • the transcription factor is selected from Onecut1, Pou4f2, and Islet1, and combinations thereof.
  • the transcription factor is selected from Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof.
  • the RGC transcription factor is Onecut1.
  • the RGC transcription factor comprises Pou4f2 and/or Islet1.
  • the RGC transcription factor comprises Irx2 and/or Neurod2.
  • the nucleic acid molecule further comprises a nucleic sequence that encodes a proneural basic helix-loop-helix (bHLH) transcription factor, as described herein.
  • the nucleic acid molecule comprises a first nucleic acid molecule encoding a proneural bHLH transcription factor, and a second nucleic acid molecule encoding a RGC transcription factor.
  • the nucleic acid molecule is administered in the form of a composition.
  • the administering comprises a first administration of composition comprising a first nucleic acid molecule encoding a proneural bHLH transcription factor, and a second administration at a subsequent time point of a composition comprising a second nucleic acid molecule encoding a RGC transcription factor selected from the group consisting of Onecut1, Pou4f2, Islet1, Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof.
  • the transcription factor is selected from Onecut1, Pou4f2, and Islet1, and combinations thereof.
  • the transcription factor is selected from Irx2, Irx5, Neurod2, Ebf1, and Tcf3, and combinations thereof.
  • the RGC transcription factor is Onecut1.
  • the RGC transcription factor comprises Pou4f2 and/or Islet1. In some embodiments, the RGC transcription factor comprises Irx2 and/or Neurod2. In some embodiments, the proneural bHLH transcription factor and the RGC transcription factor are administered as a fusion protein. In some embodiments, the first and/or second nucleic acid molecule is an mRNA. [0016] In additional embodiments, the nucleic acid molecules and methods disclosed herein stimulate production of functional RGCs from reprogrammed MG. In another embodiment, the number of the MG-derived functional RGCs is increased. In another embodiment, the number of functional RGCs is increased by 40%. In another embodiment, the subject is treated for retinal disease, damage or degeneration in the retina.
  • a vector comprises the nucleic acid molecule.
  • the vector is a non-viral vector or a viral vector, and the viral vector is an adeno-associated viral (AAV) vector or a lentiviral vector.
  • AAV adeno-associated viral
  • a promoter sequence is in operable linkage with the nucleic acid encoding the developmental RGC transcription factor.
  • the promoter is a Cre- inducible or tTA-inducible or MG-specific promoter.
  • administering to the retina is intravitreal or subretinal injection. [0017] Also provided herein are methods for inducing retinal regeneration comprising administering to a subject a composition as described herein.
  • the methods are effective to increase the number of Müller glial-derived RGCs, to induce Müller glial cells to enter the mitotic cell cycle, and/or to generate new RGCs.
  • the number of RGCs increases by at least 40% relative to a baseline level or other reference amount representative of an untreated retina.
  • the number of RGCs increases by 10%, 20%, 25%, 50%, 100%, 150%, 200%, or more.
  • the subject in the methods disclosed herein is typically a mammal, such as a human or veterinary subject. In one embodiment, the subject is an adult.
  • the subject in some embodiments, has a retinal degenerative disease.
  • retinal degenerative diseases include, but are not limited to, Age-related macular degeneration, glaucoma, ischemia, central retinal arterial occlusion and inherited retinal diseases, such as Retinitis Pigmentosa or Usher's syndrome.
  • Reprogramming of MG and regeneration of retinal neurons is particularly important for developing therapeutic products and methods for a range of degenerative ocular diseases such as, for example, and without limitation, retinal degeneration caused by diabetic retinopathy, glaucoma, and age-related macular degeneration.
  • One such retinal degenerative disease is known as central retinal artery occlusion (CRAO), wherein blood flow through the central retinal artery is blocked or occluded often resulting in loss of vision.
  • CRAO central retinal artery occlusion
  • FIGS.1A-1H Pou4f2 and/or Islet1 stimulate regeneration of RGC-like neurons.
  • Pou4f2/Islet1 is surrounded by mutually exclusive floxed sites, leading to expression of Pou4f2, Islet1, or both in the presence of active Cre.
  • FIGS.2A-2F Islet1 + Pou4f2 + Ascl1 (IPA)-stimulated MG-derived neurons display complex neuronal morphology.
  • A Retinal whole mounts stained for GFP (MG-derived cells) and Brn3.
  • FIGS.3A-3G scRNA-seq analysis of Pou4f2/Islet1-stimulated neurons reveals molecular characteristics of RGCs.
  • A UMAP plot for FACS-sorted MG-derived cells after the IPA regeneration paradigm combined with a previous scRNA-seq dataset where Ascl1 only was used.
  • C The distribution of cells from each treatment projected onto a split UMAP plot. Donut plots represent the percent each cluster comprises of the dataset.
  • D Heatmap comparing scRNA-seq datasets of Ascl1 only versus IPA treatment. Selected genes are depicted that are associated with RGCs.
  • FIGS.4A-4M Islet1 and Pou4f2 coinduction stimulates RGC-like neurons from MG in vitro.
  • A Schematic of transgenic construct to induce IPA in all primary MG in vitro by doxycycline.
  • C to E Representative images of EdU+ MG-derived neurons expressing neuronal markers.
  • C EdU+ (white) MG-derived cell expressing Tuj1 (red).
  • D MG-derived neuron expressing EdU (white), Neurofilament M (NFM; red), and the GFP transgene reporter (GFP).
  • E MG- derived neuron expressing Calbindin (red) colabeling with EdU (white), DAPI, and GFP.
  • FIGS.5A-5D Physiological profiling of IPA-induced neurons.
  • A Summary of electrical properties of cells in this study compared to endogenous neurons, endogenous glia, and MG-derived neurons from previous regeneration protocols (4, 5, 13). Resting potential and input resistance were estimated from current clamp recordings.
  • B Examples of responses to current (left) and voltage (right) steps for three cells.
  • C Three examples of cells that responded to light stimulus.
  • D Examples of cells that displayed action potentials or similar events. The two left panels are responses to hyperpolarizing and depolarizing current steps from a cell that generated apparent Na+ spikes. The right two panels are responses from a cell that generated smaller discrete events, likely Ca2+ spikes.
  • FIGS.6A-6J scATAC reveals MG remodel chromatin to an RGC-like state in response to IPA treatment.
  • A Combined UMAP of GFP+ sorted MG and their progeny from the in vivo regeneration paradigm with Ascl1-only (B) or IPA treatment (C).
  • D Coverage plots for known marker genes used to identify clusters.
  • E and F chromVAR scores of Otx2 and Pou4f2 to highlight differential accessibility of their respective motifs.
  • G Scatterplot comparing accessible motifs in E14 RGCs versus IPA-induced RGCs.
  • FIGS.7A-7I Top “GO biological process” results for peaks specific to E14 RGCs compared to IPA-derived RGC- like neurons.
  • K Retinal sections showing GFP+ MG-derived cells costained with the MG nuclei marker Sox2 (red) and quantification of GFP+ cells expressing Sox2. Scale bars, 50 ⁇ m. [0026] FIGS.7A-7I.
  • Atoh1 to the IPA paradigm facilitates transition from a progenitor state to a differentiated neuron.
  • A Schematic of transgenic construct to express IPA with Atoh1 in MG.
  • B Regeneration paradigm for inducing IPA:Atoh1 expression in MG in the damaged retina.
  • C Representative immunofluorescence images of regenerated neurons from IPA:Atoh1 mice demonstrating MG-derived neurons (GFP+) are HuC/D+ and not Otx2+.
  • D Integrated UMAP of FACS-sorted MG-derived cells after regeneration paradigm with either IPA:Atoh1 or IPA-only overexpression. Highlighted are the RGC-like cells from each dataset that were subsetted for further comparative analysis.
  • E Scatterplot highlighting differentially expressed genes between the RGC-like cells of the IPA:Atoh1 and IPA-only regeneration paradigms.
  • F GO analysis revealed that neurodevelopmental terms containing many retinal progenitor genes were down-regulated in the IPA:Atoh1 dataset versus IPA only.
  • G Integrated UMAP of IPA:Atoh1 data as described above with previously generated Ascl1:Atoh1 dataset (13). Highlighting denotes RGC-like cells from each dataset compared in further analysis.
  • H Scatterplot highlighting differentially expressed genes between the RGC-like cells of the IPA:Atoh1 and Ascl1:Atoh1 regeneration paradigms.
  • FIGS.8A-8C A subset of IPA-derived neurons are derived from proliferating MG.
  • 8A Experimental paradigm to label diving cells during the regeneration experiment described in Figure 1.
  • 8B Representative sections showing MG-derived cells (GFP+) that previously underwent cell division (EdU+).
  • 8C Representative image showing some MG- derived neurons (GFP+/HuC/D+, upper panels) are the result of proliferating MG (EdU+ lower left panel). Scale bars are 50 ⁇ m.
  • FIGS.9A-9E IPA-treatment is most effective at reprogramming MG if induced prior to injury.
  • FIGS.10A-10E scRNA-seq analysis showing Pou4f2 biases MG-production towards RGC-like neurons.
  • FIGS.11A-11D MG-derived RGCs are a stable population over time.
  • (11A) Combined UMAP of IPA-treated MG from a three and six week end point.
  • (11B) Split UMAP showing the distribution of cells in the UMAP in (a) from each time point.
  • (11C) Stacked bar graph showing the percentages of each cluster of MG and MG-derived neurons from the three week and six week time point.
  • FIGS.12A-12E MG from IPA mice express reprogramming factors.
  • FIGS.13A-13F scATAC-seq of the E14 embryonic mouse retina.
  • 13A UMAP plot of scATAC-seq from E14 embryonic mouse retina.
  • Chromvar scores show the motif accessibility used to identify the clusters of progenitors (13B, Sox2), retinal ganglion cells (13C, Pou4f2), cones (13D, Otx2), and neurogenic precursors (13E, Ascl1).
  • 13F Pseudotime subset of the transition of retinal progenitor cells to retinal ganglion cells that is further analyzed in Fig 6.
  • FIGS.14A-14H The addition of Atoh1 to IPA significantly induces MG-derived RGC- like cells and does not require retinal damage.
  • 14A Transgenic mouse construct used for induction of Ascl1, Atoh1, Pou4f2, and Islet1.
  • FIG.15 Promotion of RGC production using transcription factors specific to developing retinal cells. Lentiviruses were used to induce the expression of several transcription factors that are expressed in developing retinal cells, but not in Müller glia. The Müller glia were grown in cell culture and infected with the viruses. The cells were then cultured for 5 – 7 days and subsequently processed for single cell RNAseq to determine their fates. Of all the factors tested, Onecut1 (arrow) was able to induce the Müller glia to generate new cells with the characteristic gene expression of RGCs. [0035] FIG.16. Promotion of RGC production using transcription factors specific to developing retinal cells.
  • FIGS.17A-17C AAV delivered reprogramming transcription factors can induce neurogenesis. HuC/D+ neurons lineage was traced from MG by tdTomato.
  • 17A AAV design using Atoh1.
  • 17B Protocol for lineage tracing.
  • 17C Immunofluorescent demonstration of successful delivery of reprogramming transcription factors resulting in HuC/D+ neurons reprogrammed from MG cells.
  • FIG.18 Reprogramming efficiency of lineage-traced MG.
  • FIG.19 Irx2 and Neurod2 promote axon growth in IPA reprogrammed MG.
  • FIGS.20A-20D Human Muller glia generated in vitro from fetal retina or pluripotent stem cells using retinospheres and retinal organoids. (20A) Schematic of Embryonic Stem cell (ESC) differentiation protocol to generate retinal organoids (RO).
  • ESC Embryonic Stem cell
  • FIGS.21A-21B Protocol to generate dissociated human Muller glia cultures in vitro.
  • FIGS.22A-22E Ascl1 promotes neurogenesis in human Muller glia.
  • 22A Protocol for delivery of GFP into dissociated cells.
  • FIG.23 Characterization of ShH10 capsid and RLBP promoter in NHP dissociated Muller glia culture.
  • FIG.24 HES1 promoter as an alternative to RLBP1 promoter. Schematic illustration of a lentiviral construct containing HES1 promoter driving the expression of EGFP.
  • FIGS.25A-25D Schematic illustration of the construction of a lentivirus containing the HES1 promoter driving the expression of EGFP (control) or driving the expression of the proneural factor ASCL1 and EGFP in Muller glia (25A).
  • FIG.26 HES1-promoter also directs expression in MG in adult NHP dissociated cultures.
  • DETAILED DESCRIPTION The molecules, compositions, and methods described herein are based on the surprising discovery that retinal ganglion–like cells can be regenerated in the damaged adult retina in vivo with targeted overexpression of developmental retinal ganglion cell transcription factors. As demonstrated herein, AAV vectors can deliver reprogramming transcription factors to Muller glia in vivo.
  • Muller glia can be reprogrammed to generate neurons in dissociated cultures, and these human Muller glia can be derived from either retinal organoids or fetal human retina. This ability to reprogram Muller glia into specific types is particularly important for endogenous regeneration strategies because most blinding diseases are the result of deficits in a particular neuronal subtype. For example, glaucoma is primarily caused by the death of RGCs.
  • retina neuron refers to any of the five types of neurons in the retina: photoreceptors, bipolar cells, ganglion cells, horizontal cells, and amacrine cells. In some particular embodiments, the retinal neurons are bipolar neurons, amacrine, horizontal, and ganglion cells.
  • nucleic acid sequence or “polynucleotide” refers to nucleotides of any length which are deoxynucleotides (i.e. DNAs), or derivatives thereof; ribonucleotides (i.e. RNAs) or derivatives thereof; or peptide nucleic acids (PNAs) or derivatives thereof.
  • the terms include, without limitation, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, oligonucleotides (oligos), or other natural, synthetic, modified, mutated or non-natural forms of DNA or RNA.
  • MicroRNAs or “miRNAs”, or “miRs”, are short, non-coding RNAs that regulate gene expression by post-transcriptional regulation of target genes.
  • “Short hairpin RNAs” or “shRNAs” are synthetic or non-natural RNA molecules.
  • shRNA refers to RNA with a tight hairpin turn used to silence (via RNA interference or RNAi) target gene expression in a cell.
  • An shRNA is typically delivered via an expression vector such as a DNA plasmid or via viral vectors.
  • the term “vector” refers to, without limitation, a recombinant genetic construct or plasmid or expression construct or expression vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and integrate into the target cell’s genome.
  • the vector may be derived from or based on a wild-type virus. Aspects of this disclosure relate to an adeno-associated virus vector, an adenovirus vector, and a lentivirus vector.
  • expression control element refers to any sequence that regulates the expression of a coding sequence, such as a gene.
  • Exemplary expression control elements include but are not limited to promoters, enhancers, microRNAs, post- transcriptional regulatory elements, polyadenylation signal sequences, and introns.
  • Expression control elements may be, without limitation, constitutive, inducible, repressible, or tissue-specific.
  • a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • expression control by a promoter is tissue-specific.
  • An “enhancer” is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription.
  • Non-limiting exemplary enhancers and posttranscriptional regulatory elements include the CMV enhancer and WPRE.
  • multicistronic or “polycistronic” or “bicistronic” or tricistronic” refers to mRNA with multiple, i.e., double or triple coding areas or exons, and as such will have the capability to express from mRNA two or more, or three or more, or four or more, etc., proteins from a single construct. Multicistronic vectors simultaneously express two or more separate proteins from the same mRNA.
  • the two strategies most widely used for constructing multicistronic configurations are through the use of 1) an IRES or 2) a 2A self- cleaving site.
  • an “IRES” refers to an internal ribosome entry site or portion thereof of viral, prokaryotic, or eukaryotic origin which are used within polycistronic vector constructs.
  • an IRES is an RNA element that allows for translation initiation in a cap- independent manner.
  • self-cleaving peptides or “sequences encoding self- cleaving peptides” or “2A self-cleaving site” refer to linking sequences which are used within vector constructs to incorporate sites to promote ribosomal skipping and thus to generate two polypeptides from a single promoter, such self-cleaving peptides include without limitation, T2A, and P2A peptides or sequences encoding the self-cleaving peptides.
  • substantially complementary when used to define either amino acid or nucleic acid sequences, means that a particular sequence, for example, an oligonucleotide sequence, is substantially complementary to the sequence referenced.
  • sequences will be highly complementary to the “target” sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence.
  • highly complementary sequences will typically bind quite specifically to the target sequence region and will therefore be highly efficient in reducing, and/or even inhibiting the biological activity of the target sequence.
  • Substantially complementary nucleic acid sequences will be greater than about 80 percent complementary (or ‘% exact-match’) to the corresponding target sequence to which the nucleic acid specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds.
  • nucleic acid sequences will be greater than about 90 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including 96%, 97%, 98%, 99%, and even 100% exact match complementary to the target to which the designed nucleic acid specifically binds.
  • “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules.
  • Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of disclosed herein. [0060] Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • UWGCG University of Wisconsin Genetics Computer Group
  • the GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non- identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • Nucleotide sequence refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of specifically binding to a target molecule and acting as an antisense construct to alter, reduce, or inhibit the biological activity of the target.
  • “directed against”, in the context of antisense oligonucleotides, means the antisense oligonucleotide binds to a target miRNA and blocks or suppresses activity of the target.
  • the terms “protein”, “peptide”, and “polypeptide” refer to amino acid subunits, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids.
  • the term “recombinant expression system” or “recombinant expression vector” refers to a genetic construct for the expression of certain genetic material formed by recombination.
  • the term “effective amount” or “therapeutically effective amount” or “prophylactically effective amount”, refer to an amount of an active agent described herein that is effective to provide the desired/intended result and/or biological activity.
  • an effective amount of a composition described herein is an amount that is effective to result in regeneration of retinal neurons, and/or to improve or to ameliorate symptoms of and/or to treat retinal degenerative diseases.
  • the disclosure herein relates to a small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense, or miRNA
  • an equivalent or a biologically equivalent of such is intended within the scope of this disclosure.
  • biological equivalent thereof is intended to be synonymous with “equivalent thereof” when referring to a reference small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense, or miRNA even those reference molecules having minimal homology while still maintaining desired structure or functionality.
  • any nucleic acid, polynucleotide, oligonucleotide, antisense, miRNA, polypeptide, or protein mentioned herein also includes equivalents thereof.
  • an equivalent intends at least about 70% homology or identity, or at least 80% homology or identity and alternatively, or at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
  • polypeptide and/or polynucleotide sequences are provided herein for use in gene and protein transfer and expression techniques described below. Such sequences provided herein can be used to provide the expression product as well as substantially identical sequences that produce a protein that has the same biological properties. These “biologically equivalent” or “biologically active” or “equivalent” polypeptides are encoded by equivalent polynucleotides as described herein.
  • They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical primary amino acid sequence to the reference polypeptide when compared using sequence identity methods run under default conditions.
  • Specific polynucleotide or polypeptide sequences are provided as examples of particular embodiments. Modifications may be made to the amino acid sequences by using alternate amino acids that have similar charge.
  • an equivalent polynucleotide is one that hybridizes under stringent conditions to the reference polynucleotide or its complement or in reference to a polypeptide, a polypeptide encoded by a polynucleotide that hybridizes to the reference encoding polynucleotide under stringent conditions or its complementary strand.
  • an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.
  • “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, 0.1x SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • treating or “treatment” of a retinal degenerative disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms of retinal degeneration, diminishment of extent of a retinal degenerative condition (including a retinal degenerative disease), stabilized (i.e., not worsening) state of a retinal degenerative condition (including disease), delay or slowing of a retinal degenerative condition (including disease), progression, amelioration or palliation of a retinal degenerative condition (including disease), states of and remission of (whether partial or total) retinal degeneration, whether detectable or undetectable.
  • the term "isolated” means that a naturally occurring DNA fragment, DNA molecule, coding sequence, or oligonucleotide is removed from its natural environment, or is a synthetic molecule or cloned product.
  • the DNA fragment, DNA molecule, coding sequence, or oligonucleotide is purified, i.e., essentially free from any other DNA fragment, DNA molecule, coding sequence, or oligonucleotide and associated cellular products or other impurities.
  • the term “cell” as used herein refers to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.
  • Cells treated, transfected, transformed, or otherwise in contact with compositions and/or nucleic acid molecules disclosed herein include without limitation, cells of a human, non-human animal, mammal, or non-human mammal, including without limitation, cells of murine, canine, or non-human primate species.
  • Cells treated, transfected, transformed, or otherwise in contact with compositions and/or nucleic acid molecules disclosed herein are, without limitation, retinal cells, Müller glia (MG), and/or retinal neuronal cells such as retinal neurons, bipolar neurons, amacrine cells, horizontal cells, ganglion cells and/or glia.
  • Müller glial cells or “Müller glia” or “MG” refer to cells which are found in the vertebrate retina and are support cells for neurons. MG are the most common type of glial cells in the retina. While MG cell bodies are located in the inner nuclear layer of the retina, MG span across the entire retina. [0073] As used herein, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects.
  • encode as it is applied to nucleic acid sequences refers to a polynucleotide which is said to “encode” a polypeptide, an mRNA, or an effector RNA if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce the effector RNA, the mRNA, or an mRNA that can for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • expression or “gene expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • the expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample; further, the expression level of multiple genes can be determined to establish an expression profile for a particular sample.
  • the term “functional” may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect.
  • the term “combined therapy” refers to two or more compositions and/or nucleic acid molecules, delivered in combination, for example and without limitation, sequentially, concurrently, simultaneously, and/or step-wise, in order to achieve a therapeutic effect.
  • enhancing expression levels of the two or more proneural bHLH transcription factors, endogenous and/or exogenous refers to an increase in the amount of expressed as compared to a control sample or explant levels of endogenous and/or exogenous Ascl1, and/or Atoh1, and/or Atoh7 such as, without limitation, untreated, or Ascl1 expression alone.
  • neurogenesis is increased and/or the production of functional neurons is increased as compared to a control.
  • expression levels and/or functional neurons are increased about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 20 fold, about 50 fold, about 100 fold, about 1000 fold, or about 10,000 fold relative to the control.
  • reprogramming potentiator or “reprogramming potentiating agent”, used herein interchangeably, refers to a small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense, miRNA, or an equivalent or a biologically equivalent thereof which assists in the process of stimulating and/or boosting neurogenesis from MG in a manner such that functional neurons from the MG are produced.
  • one or more reprogramming potentiators assist in the process of stimulating neurogenesis and producing functional neurons from MG by inhibiting the HDAC pathway.
  • one or more reprogramming potentiators assist in the process of stimulating neurogenesis and producing functional neurons from MG by inhibiting the Jak/STAT pathway. In another embodiment, one or more reprogramming potentiators assist in the process of stimulating neurogenesis and producing functional neurons from MG by inhibiting the HDAC + Jak/STAT pathways. In another embodiment, one or more reprogramming potentiators assist in the process of stimulating neurogenesis and producing functional neurons from MG by enhancing and/or increasing endogenous and/or exogenous Ascl1 expression levels. See also our previous work in WO2019/210320 and WO2020/223308, each of which is incorporated herein by reference in its entirety.
  • AAV adeno-associated virus
  • Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 or 12 serotypes, e.g., AAV2, AAV5, and AAV8, or engineered serotypes, e.g. AAV-SHH10 and AAV-7m8.
  • the AAV structural particle is composed of 60 protein molecules made up of VP1, VP2, and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins and 50 VP3 proteins ordered into an icosahedral structure.
  • nucleic Acid Molecules and Compositions are provided for retinal regeneration, the potentiation of retinal regeneration, stimulation of regeneration of retinal ganglion cells, restoration of vision, and for treatment of retinal degenerative disease, damage, or injury.
  • Such nucleic acid molecules may be delivered by viral or non-viral means.
  • viral delivery is adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • retrovirus and lentivirus delivery include retrovirus and lentivirus delivery.
  • CPP cell penetrating peptide
  • Polynucleotide constructs may also be modified, such as through chemical modification, to improve their stability and/or suitability for delivery.
  • the oligonucleotide is modified by locked nucleic acids and/or phosphorothioate linkages.
  • a delivery system is selected for improved bioavailability, such as PEGylated liposomes, lipidoids, or biodegradable polymers, as examples.
  • the composition further comprises one or more additional potentiating or therapeutic agents, including, for example, reprogramming potentiating agents.
  • the composition is free of reprogramming potentiating agents.
  • a composition comprising one or more small molecule reprogramming potentiating agents can be administered sequentially or concurrently with the nucleic acid molecules disclosed herein.
  • one or more protein/peptide or miR- based reprogramming potentiators can be incorporated into the nucleic acid molecules disclosed herein.
  • Such one or more reprogramming potentiators are selected from HDACi, STATi, Jak/STATi and RNAi-based Ascl1 activators. See also our previous work in WO2019/210320 and WO2020/223308, each of which is incorporated herein by reference in its entirety.
  • the HDAC signaling pathway inhibitor is selected from the group consisting of peptidomimetics, small molecule inhibitors, oligonucleotides, peptides and proteins.
  • HDACi small molecule HDACi
  • TSA trichostatin A
  • Istodax TM also known as (Pro)/romidepsin
  • Beleodaq TM also known as (Pro)/belinostat
  • Farydak TM also known as (Pro)/panobinostat
  • Zolinza TM also known as (Pro)/vorinostat
  • Quisinostat Abexinostat, Givinostat, Resminostat, Phenylbutyrate
  • Valproic Acid Depsipeptide
  • Entinostat Mocetinostat
  • Tubastatin A tubastatin A.
  • Exemplary HDACi peptides are, without limitation, 16cyc-HxA, 16lin-HxA and 16KA.
  • the inhibitor, mimic, activator, or antagomir is an oligonucleotide or a nucleotide sequence.
  • the invention thus provides nucleotide constructs for use in the compositions or combined therapy or nucleic acid molecules and methods described herein.
  • the reprogramming potentiating agents are selected from one or more STAT signaling pathway inhibitors; and one or more Ascl activators such as, without limitation, miR-25 and/or miR-124; and one or more let-7 family inhibitors.
  • composition comprising any one or more of the combined therapy of RNAi-based Ascl1 activators and/or HDACi + STATi, and/or a nucleic acid sequence encoding the developmental RGC transcription factors or a vector comprising the nucleic acid sequences disclosed herein, and a carrier.
  • the carrier is a pharmaceutically acceptable carrier.
  • An exemplary nucleic acid sequence encoding human Onecut1 can be found at NCBI Reference Sequence number NC_000015.10 (SEQ ID NO: 1).
  • An exemplary nucleic acid sequence encoding human Pou4f2 can be found at NCBI Reference Sequence number NC_000004.12 (SEQ ID NO: 3).
  • An exemplary nucleic acid sequence encoding human Islet1 can be found at NCBI Reference Sequence number NC_000005.10 (SEQ ID NO: 5).
  • An exemplary nucleic acid sequence encoding human Irx2 can be found at NCBI Reference Sequence number NC_000005.10 (SEQ ID NO: 8).
  • the mouse version of Irx2 used in the Examples herein can be found at NCBI Reference Sequence: NM_010574.4 (SEQ ID NO: 64; encoding the amino acid sequence of SEQ ID NO: 65).
  • An exemplary nucleic acid sequence encoding human Irx5 can be found at NCBI Reference Sequence number NC_000016.10 (SEQ ID NO: 13) .
  • the mouse version of Irx5 used in the Examples herein can be found at NCBI Reference Sequence: NM_018826.2 (SEQ ID NO: 66; encoding the amino acid sequence of SEQ ID NO: 67).
  • An exemplary nucleic acid sequence encoding Neurod2 can be found at NCBI Reference Sequence number NC_000017.11 (SEQ ID NO: 17).
  • An exemplary nucleic acid sequence encoding human Ebf1 can be found at NCBI Reference Sequence number NC_000005.10 (SEQ ID NO: 19).
  • the mouse version of Ebf1 used in the Examples herein can be found at NCBI Reference Sequence: NM_001290709.1 (SEQ ID NO: 68; encoding the amino acid sequence of SEQ ID NO: 69).
  • An exemplary nucleic acid sequence encoding human Tcf3 can be found at NCBI Reference Sequence number NC_000019.10 (SEQ ID NO: 48).
  • the mouse version of Tcf3 used in the Examples herein can be found at NCBI Reference Sequence: NM_001164147.2 (SEQ ID NO: 70; encoding the amino acid sequence of SEQ ID NO: 71). This mouse sequence can be used to guide the selection of a corresponding human or humanized Tcf3.
  • An exemplary nucleic acid sequence encoding human Ascl1 can be found at NCBI Reference Sequence number NG_008950.1 (SEQ ID NO: 50).
  • nucleic acid sequence encoding a human Ascl1 amino acid sequence or portion thereof of UniProtKB/Swiss-Prot: P50553.2 (SEQ ID NO: 51).
  • Ascl1 homologues e.g., derived from species such as murine, canine, equine, are included herein, without limitation.
  • the Protein Atonal Homolog 1 (Atoh1) is a proneural member of the family of bHLH transcription factors. The protein activates a different E box than the Ascl1 gene.
  • An exemplary nucleic acid sequence encoding human Atoh1 can be found at NCBI Reference Sequence number NM_005172.1 (SEQ ID NO: 52).
  • Atoh1 amino acid sequence or portion thereof of NP_005163.1 (SEQ ID NO: 53). Atoh1 homologs, orthologs and/or paralogs, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.
  • the Atoh7 family bHLH transcription factor 7 (Atoh7) gene encodes a proneural member of the basic helix-loop-helix (BHLH) family of transcription factors.
  • An exemplary nucleic acid sequence encoding human Atoh7 can be found at NCBI Reference Sequence number NM_008553.4 (SEQ ID NO: 54).
  • nucleic acid sequence encoding a human Atoh7 amino acid sequence or portion thereof of NP_660161.1 (SEQ ID NO: 55). Atoh7 homologs, orthologs, and/ paralogs, e.g., derived from species such as murine, canine, equine, are included herein, without limitation.
  • An exemplary nucleic acid sequence encoding Neurogenin-2 (also known as NEUROG2 and NGN-2) can be found at NCBI Reference Sequence number NM_024019.
  • An exemplary nucleic acid sequence encoding Neurod1 can be found at NCBI Reference Sequence number KR709666.
  • Exemplary nucleic acid sequences of the Ascl1, Atoh1, and/or Atoh7 or other proneural bHLH transcription factor for use herein include, without limitation, portions thereof of the corresponding sequences of Ascl1, Atoh1, and/or Atoh7, for the purposes of configurating multicistronic, bicistronic, and/or tricistronic constructs, plasmids, and/or expression vectors. It is understood that portions of the sequences referenced herein can be selected for use, wherein the selected portions are sufficient to encode the recited transcription factor(s) and/or other elements.
  • the vector disclosed herein is a viral vector.
  • the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector.
  • the vector is a retroviral vector, an adenoviral/retroviral chimera vector, a herpes simplex viral I or II vector, a parvoviral vector, a reticuloendotheliosis viral vector, a polioviral vector, a papillomaviral vector, a vaccinia viral vector, or any hybrid or chimeric vector incorporating favorable aspects of two or more viral vectors.
  • the vector further comprises one or more expression control elements operably linked to the polynucleotide.
  • the vector further comprises one or more selectable markers.
  • the vector disclosed herein is an AAV vector with low toxicity.
  • the AAV vector does not incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis.
  • the AAV vector can encode a range of total polynucleotides from 4.5 kb to 4.75 kb.
  • exemplary AAV vectors that may be used in any of the herein described compositions, systems, methods, and kits can include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32/33 vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector and any combinations or equivalents thereof.
  • the vector disclosed herein is a lentiviral vector.
  • the lentiviral vector is an integrase-competent lentiviral vector (ICLV).
  • the lentiviral vector can refer to the transgene plasmid vector as well as the transgene plasmid vector in conjunction with related plasmids (e.g., a packaging plasmid, a rev expressing plasmid, an envelope plasmid) as well as a lentiviral-based particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • Lentiviral vectors are well-known in the art.
  • exemplary lentiviral vectors that may be used in relation to any of the herein described compositions, nucleic acid molecules and/or methods, and can include a human immunodeficiency virus (HIV) 1 vector, a modified human immunodeficiency virus (HIV) 1 vector, a human immunodeficiency virus (HIV) 2 vector, a modified human immunodeficiency virus (HIV) 2 vector, a sooty mangabey simian immunodeficiency virus (SIV SM ) vector, a modified sooty mangabey simian immunodeficiency virus (SIV SM ) vector, a African green monkey simian immunodeficiency virus (SIV AGM ) vector, a modified African green monkey simian immunodeficiency virus (SIV AGM ) vector, a equine infectious anemia virus (EIAV) vector, a modified equine infectious anemia virus (EIAV) vector, a feline immunode
  • HAV
  • a vector of the disclosure is a viral vector.
  • the viral vector comprises a sequence isolated or derived from a retrovirus.
  • the viral vector comprises a sequence isolated or derived from a lentivirus.
  • the viral vector comprises a sequence isolated or derived from an adenovirus.
  • the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the viral vector is replication incompetent.
  • the viral vector is isolated or recombinant.
  • the viral vector is self-complementary.
  • the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • the viral vector comprises an inverted terminal repeat sequence or a capsid sequence that is isolated or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or the vector and/or components are derived from a synthetic AAV serotype, such as, without limitation, Anc80 AAV (an ancestor of AAV 1, 2, 6, 8 and 9).
  • the viral vector is replication incompetent.
  • the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self- complementary (scAAV).
  • a vector of the disclosure is a non-viral vector. In some embodiments, the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex or a dendrimer.
  • expression vector or viral vector disclosed herein is used to transfect, transform, or come in contact with a cell which is a eukaryotic cell. In some embodiments, the cell is an animal cell.
  • the cell is a mammalian cell.
  • the cell is a bovine, murine, feline, equine, porcine, canine, simian, or human cell.
  • the cell is a retinal neuron or MG of an animal or mammal.
  • a cell is a packaging cell or a producer cell for production of a viral particle.
  • viral particles comprising, consisting of, or consisting essentially of a vector comprising, consisting of, or consisting essentially of a polynucleotide sequence encoding a developmental RGC transcription factor and, optionally, an Ascl1 protein.
  • the packaging vector may include, but is not limited to retroviral vector, lentiviral vector, adenoviral vector, and adeno-associated viral vector.
  • the packaging vector contains elements and sequences that facilitate the delivery of genetic materials into cells.
  • the retroviral constructs are packaging plasmids comprising at least one retroviral helper DNA sequence derived from a replication- incompetent retroviral genome encoding in trans all virion proteins required to package a replication incompetent retroviral vector, and for producing virion proteins capable of packaging the replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus.
  • the retroviral DNA sequence lacks the region encoding the native enhancer and/or promoter of the viral 5’ LTR of the virus, and lacks both the psi function sequence responsible for packaging helper genome and the 3’ LTR, but encodes a foreign polyadenylation site, for example the SV40 polyadenylation site, and a foreign enhancer and/or promoter which directs efficient transcription in a cell type where virus production is desired.
  • the retrovirus is a leukemia virus such as a Moloney Murine Leukemia Virus (MMLV), the Human Immunodeficiency Virus (HIV), or the Gibbon Ape Leukemia virus (GALV).
  • the foreign enhancer and promoter may be the human cytomegalovirus (HCMV) immediate early (IE) enhancer and promoter, the enhancer and promoter (U3 region) of the Moloney Murine Sarcoma Virus (MMSV), the U3 region of Rous Sarcoma Virus (RSV), the U3 region of Spleen Focus Forming Virus (SFFV), or the HCMV IE enhancer joined to the native Moloney Murine Leukemia Virus (MMLV) promoter.
  • HCMV human cytomegalovirus
  • IE immediate early
  • IE Enhancr and promoter
  • U3 region of the Moloney Murine Sarcoma Virus
  • RSV Rous Sarcoma Virus
  • SFFV Spleen Focus Forming Virus
  • HCMV IE enhancer joined to the native Moloney Murine Leukemia Virus
  • the retroviral packaging plasmid may consist of two retroviral helper DNA sequences encoded by plasmid-based expression vectors, for example where a first helper sequence contains a cDNA encoding the gag and pol proteins of ecotropic MMLV or GALV and a second helper sequence contains a cDNA encoding the env protein.
  • the Env gene which determines the host range, may be derived from the genes encoding xenotropic, amphotropic, ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virus env proteins, or the Gibbon Ape Leukemia Virus (GALV env protein, the Human Immunodeficiency Virus env (gp160) protein, the Vesicular Stomatitus Virus (VSV) G protein, the Human T cell leukemia (HTLV) type I and II env gene products, chimeric envelope gene derived from combinations of one or more of the above env genes or chimeric envelope genes encoding the cytoplasmic and transmembrane of the above env gene products and a monoclonal antibody directed against a specific surface molecule on a desired target cell.
  • GLV env protein Gibbon Ape Leukemia Virus
  • gp160 Human Immunodeficiency Virus env
  • VSV Vesicular Stomatitus
  • compositions disclosed herein include one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • a cell of the disclosure is a retinal cell, such as a Müller glial (MG) cell, or a rod or cone photoreceptor cell.
  • the cell is a neuronal cell.
  • a neuronal cell of the disclosure is a neuron of the retina.
  • a neuron cell of the disclosure is a neuron of an optic nerve.
  • a neuron cell of the disclosure is a neuroglial or a glial cell.
  • a cell is a bipolar neuron, a horizontal cell, a ganglion cell, or an amacrine cell.
  • a cell of the disclosure is an astrocyte.
  • cells of the disclosure are macroglia or microglia or glia.
  • a cell of the disclosure is a cultured cell.
  • a cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the cells are modified ex vivo and transplanted into and/or administered to the retina of a subject in need thereof.
  • a cell of the disclosure is autologous or allogeneic and used for transplantation.
  • a cell of the disclosure is a stem cell-derived or an embryonic stem cell-derived retinal cell.
  • the cell is derived from an induced pluripotent stem cell (iPS cell)-derived retinal cell.
  • iPS cell induced pluripotent stem cell
  • the cell is derived from a retinal organoid.
  • methods for inducing retinal regeneration comprising administering to a subject a composition as described herein.
  • the methods are effective to increase the number of Müller glial-derived retinal ganglion cells, to induce Müller glial (MG) cells to enter the mitotic cell cycle, and/or to generate new retinal neurons, including the generation of new ganglion cells.
  • the number of retinal neurons increases by at least 25% relative to a baseline level or other reference amount representative of an untreated retina.
  • the number of retinal neurons increases by at least 40%. In some embodiments, the number of retinal neurons increases by 10%, 20%, 50%, 100%, 150%, 200%, or more.
  • methods disclosed herein may utilize combined therapy compositions comprising one or more, or two or more, small molecule reprogramming potentiating agents.
  • the agents can be administered sequentially or concurrently with the nucleic acid molecules disclosed herein.
  • one or more protein/peptide or miR-based reprogramming potentiators can be incorporated into the nucleic acid molecules used in the methods disclosed herein. In some embodiments, the method is performed in the absence of such reprogramming potentiators.
  • the subject is typically a mammal, such as a human or veterinary subject. In one embodiment, the subject is an adult.
  • the subject in some embodiments, has a retinal degenerative disease. Examples of such retinal degenerative diseases include, but are not limited to, Age-related Macular Degeneration (AMD), Retinitis Pigmentosa (RP), Diabetic Retinopathy (DR), Central Retinal Artery Occlusion (CRAO), Vitreoretinopathy, and Glaucoma.
  • AMD Age-related Macular Degeneration
  • RP Retinitis Pigmentosa
  • DR Diabetic Retinopathy
  • CRAO Central Retinal Artery Occlusion
  • Vitreoretinopathy and Glaucoma.
  • Administration and Dosage [0127]
  • the compositions and/or nucleic acid molecules disclosed herein are administered in any suitable manner, often with pharmaceutically acceptable carriers.
  • Suitable methods of administering compositions, compounds, molecules, nucleic acids, and vectors in the context of the present invention to a subject’s eye or retina are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • intraocular injection such as, for example and without limitation, intravitreal injection and subretinal injection are the most common routes of delivery to the retina.
  • periocular, suprachoroidal, systemic, or topical administration is more suitable for efficacy and safety of delivery.
  • the dose administered to a patient should be sufficient to result in a beneficial therapeutic response in the patient over time, or to inhibit disease progression.
  • the composition is administered to a subject in an amount sufficient to elicit an effective response and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the retinal disease or injury.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Routes, order and/or frequency of administration of the therapeutic compositions disclosed herein, as well as dosage will vary from individual to individual, and may be readily established using standard techniques. In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • Example 1 Reprogramming Müller glia to regenerate ganglion-like cells with developmental transcription factors
  • Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system.
  • glial cells spontaneously reprogram into neural progenitors and replace neurons after injury.
  • TFs transcription factors
  • bHLH proneural basic helix-loop-helix
  • Ascl1 Ascl1
  • Ascl1-expressing MG adopt a molecular phenotype similar to developing retinal progenitors, and a subset of these cells undergoes mitotic division (5, 12). Some of these newly generated cells go on to differentiate into retinal neurons that connect with the endogenous circuitry (4, 5).
  • Ascl1 to stimulate MG neurogenesis causes most MG-derived neurons to take on a bipolar cell fate, with a minority resembling amacrine cells (4, 12). Recently, we reported that the efficiency and range of neuronal cell types generated through MG reprogramming can be substantially improved by adding an additional bHLH TF of the atonal class (Atoh1/7). With this combination, up to 80% of the MG expressing Ascl1 and Atoh1 will become neurogenic precursors and ultimately neurons (13).
  • the MG-derived RGC-like neurons (i) can be immunolabeled with markers of normal RGCs; (ii) have a transcriptome similar to developing RGCs by single-cell RNA sequencing (scRNA-seq); (iii) have a broader range of electrophysiological characteristics than neurons generated by Ascl1 alone, such as action potentials; and (iv) display a pattern of chromatin accessibility similar to developing RGCs.
  • ovomucoid (Worthington) was added.
  • Cells were then spun at 4°C at 300g for 10 min and resuspended in growth medium consisting of Neurobasal (Gibco), 10% fetal bovine serum (FBS) (Clontech), N2 (Invitrogen), 1 mM L-glutamine (Invitrogen), 1% penicillin-streptomycin (Invitrogen), and mouse epidermal growth factor (100 ng/ml) (R&D Systems).
  • FBS fetal bovine serum
  • N2 Invitrogen
  • 1 mM L-glutamine Invitrogen
  • penicillin-streptomycin Invitrogen
  • mouse epidermal growth factor 100 ng/ml
  • the (i) Glast-CreER:LNL-tTA:tetO- mAscl1-ires-GFP, (ii) Glast-CreER:LNL-tTA:tetO-P&I:tetO-mAscl1-ires-GFP, (iii) Glast- CreER:LNL-tTA:tetO-Atoh1:tetO-P&I:tetO-mAscl1-ires-GFP mice, (iv) rtTa:tetO-Ascl1-ires- GFP, and (v) rtTa:tetO-P7I:tetO-Ascl1-ires-GFP are from mixed backgrounds of C57BL/6 and B6SJF1.
  • the Glast-CreER, LNLtTA, and rtTa mice are from the Jackson Laboratory.
  • the tetO-mAscl1-GFP mice were a gift from M. Nakafuku (University of Cincinnati), the tetO- Atoh1 mice were a gift from P. Chen (Emory University), and the tetO-P&I mice were a gift from X. Mu (University of Buffalo). Males and females were both used in experiments at equal frequencies. All in vivo experiments were performed on adult mice that were over 40 days old.
  • Fluorescence-activated cell sorting Following euthanasia, retinas were dissociated into single cells as described for cell culture; after pelleting at 300g at 4°C, cells resuspended in Neurobasal solution and passed through a 35- ⁇ m filter. Using a BD FACSAria III cell sorter (BD Bioscience), FACS was performed on GFP+ cells. [0147] Injections [0148] Intravitreal injections were performed with a 32-G Hamilton syringe on mice anesthetized with isoflurane. Injections of NMDA were done in a volume of 1 ⁇ l at a concentration of 100 mM in PBS.
  • TSA (Sigma-Aldrich) was administered via intravitreal injections in DMSO at a concentration of 1 ⁇ g/ ⁇ l.
  • Intraperitoneal injections of tamoxifen (1.5 mg per 100 ⁇ l of corn oil) were administered to adult mice for four consecutive days to induce expression of the tetO-mAscl1-ires-GFP, the tetO-P&I, and the tetO-Atoh1 gene.
  • Microscopy/cell counts [0150] Images were taken on a Zeiss LSM880 confocal microscope. For quantification of cell counts, a minimum of four images per retina with a 20 ⁇ objective were taken at the same magnification.
  • Electrophysiology Recordings were performed identical to our previous reports (4, 5). Mice were dark- adapted before recordings. After euthanasia, retinas were sliced into 200- ⁇ m slices for recording. Tissue recordings were performed in Ames medium at 32° C and oxygenated with 95% O2/5% CO2. GFP+ cells were targeted for recording using video differential interference contrast with infrared light and confocal microscopy. Light responses were measured under infrared conditions, and the tissue was exposed to full-field illumination via blue and green light-emitting diodes.
  • the pellet was resuspended in culture medium to reach a targeted concentration of 1000 cells/ ⁇ l.
  • Cells were passed through a strainer and loaded into the 10x Genomics Chromium Single Cell chip G following the protocol of Chromium Single Cell 3′ Reagents Kits v3.1 (10x Genomics, Pleasanton, CA).
  • Single-cell RNA sequencing, mapping, and data analysis [0157] Libraries were sequenced using an Illumina NextSeq 500, in most cases using multiplexed libraries using high-output 150 kits. Data were demultiplexed and aligned to the mm10 genome using CellRanger version 3.0.
  • Filtered output files were further analyzed in R using Seurat version ⁇ 3.0, ggplot2, data.table, dplyr, tidyr, and other commonly used R packages.
  • Low-quality cells identified as having low read depth or high mitochondrial content; >10%) were removed from datasets.
  • gene expression data were normalized and scaled, and cells were clustered using principal components analysis and UMAP, using the tools available in the Seurat R package version ⁇ 3.0.
  • IPA neurons were integrated directly with a subset composed only of E14 cells from the development dataset.
  • Single-cell ATAC sequencing [0161] The Cellranger ATAC pipeline (2.0.0) was used to preprocess the data resulting from sequencing (47). First, “cellranger-atac mkfastq” was used to convert BCL files to fastqs and demultiplex reads. Next, “cellranger-atac count” was run to map Tn5 sites to mm10 (mouse genome), remove duplicate reads, and remove background cells. This returned peak by cell matrices and barcoded fragment files that were loaded into Signac (48), an R (4.0.4) (R core team, 2021) package.
  • Macs2 was then run on the Signac object and barcoded fragment files to call peaks using Signac’s “CallPeaks” function (49). Fragments were mapped to the peaks called by Macs2 and assigned to cells using Signac’s “FeatureMatrix” function. Further quality control (QC) metrics were measured in Signac using the “NucleosomeSignal” and “TSSEnrichment” functions. Cells who were outliers in the QC metric categories were removed as per Signac’s standard processing guidelines. Latent semantic indexing (LSI) was performed in Signac using the “RunTFIDF” and “RunSVD” functions.
  • LSI Latent semantic indexing
  • Signac’s “DepthCor” was used to identify LSI dimensions that were highly correlated with read depth; these LSI dimensions were excluded from downstream analysis. Signac/Seurat’s “RunUMAP” function was run to compute the UMAP embedding. To identify clusters, Signac/Seurat’s “FindClusters” was then run at varying resolutions. Clusters were assigned to known retinal cell types by inspecting Tn5 insertions within 100 kb of known marker genes using Signac’s “CoveragePlot” and further supported using chromVAR scores for known lineage-specific TFs. Clusters of the same type were grouped for visualization purposes. Vertebrate motifs were acquired from the Jaspar 2020 database.
  • Signac’s “AddMotifs” function was used to map these motifs to peaks within the Signac object. Signac’s “RunChromvar” function was used to calculate motif accessibility z score across all cells. [0162] Dataset integration [0163] Before integrating Signac objects, we first ran all previous computational steps on each sample independently. Next, we created a shared peak set for all objects that were to be integrated using BEDOPS (-m) (50). Signac’s FeatureMatrix function was run on each sample with the merged peak set to put all samples in the same feature space. Samples were next downsampled to the same average read depth using DropletUtils “downsampleMatrix” function.
  • Variable motifs were identified by running chromVAR’s “addGCBias,” “getBackgroundPeaks,” “computeDeviations,” and “computeVariability” functions. Peaks with a verbality score greater than 1.2 were kept for further analysis. This motif list was then subset again; only motifs corresponding to TFs expressed by more than 10% of cells in the RGC branch were kept. Last, conjoined motifs were dropped. Motif enrichment scores for selected motifs were ordered over pseudotime in the E14 RGC branch in the scATAC-seq data by fitting their ChomVAR scores to a third-order polynomial function and ordering motifs by the maximum value of this function within our pseudotime range.
  • chromVAR motif accessibility z scores were plotted in the previously derived order over pseudotime within the RGC lineage.
  • RNA heatmaps were created by plotting the genes whose binding sites were in the final motif list in the same order across pseudotime in the RNA object within RGC lineage.
  • the cascade heatmap for the reprogrammed cells was created by plotting motifs and factors identified in the developmental data.
  • the tetO-IPA mouse line allows us to test whether MG reprogramming is enhanced by treatment with Pou4f2 + Ascl1, Islet1 + Ascl1, or Islet1 + Pou4f2 + Ascl1 (hereafter IPA).
  • IPA a genetic polymerase chain containing protein
  • FIG. 1 shows that this protocol induces the transgenes in MG and neurons derived from them [green fluorescent protein positive (GFP+)].
  • GFP+ green fluorescent protein positive
  • IHC immunohistochemistry
  • MG-derived GFP+ neuronal-like cells expressed the ganglion/amacrine marker HuC/D (Fig.1, F and G) or the bipolar marker Otx2 (Fig.1, G and H).
  • IPA expression substantially enhanced MG neurogenesis of HuC/D neurons compared to Ascl1 alone (Fig.1G). Consistent with our previous reports, a subset of the MG-derived neurons was derived from EdU+, proliferating MG (fig.8A-8C).
  • IPA induces neurons with an RGC-like transcriptome
  • scRNA-seq we next used to analyze how Islet1 and Pou4f2 alter the phenotype of Ascl1-mediated MG reprogramming. Three weeks after initiating the IPA regeneration protocol, MG cells and their progeny were fluorescence-activated cell sorting (FACS)– purified and processed for scRNA-seq as previously described (5, 12). To directly compare the changes in cell fates induced by IPA with those caused by expression of Ascl1 alone, we used Seurat to integrate data from IPA treatment with previously obtained Ascl1-only reprogramming libraries (4) and clustered the cells (Fig.3A).
  • the combined data from the IPA experiment and the prior Ascl1 dataset were projected onto a single uniform manifold approximation and projection (UMAP) plot and clusters of cell types were identified by known marker genes (Fig.3B).
  • UMAP uniform manifold approximation and projection
  • Fig.3A The combined UMAP plot of Ascl1-only versus IPA treatment contains clusters of cell types (e.g., MG, progenitors, and bipolar cells) that we have previously observed during Ascl1-mediated reprogramming (Fig.3A). This analysis revealed two additional phenotypes unique to the IPA condition.
  • the new cluster of cells induced by IPA shows a high expression of genes characteristic of RGCs, such as Elavl4 and Sox11 (Fig.3B) (20).
  • these IPA neurons expressed many genes found in the gene ontology (GO) terms “axon guidance,” “axon outgrowth,” and “axogenesis” (fig.10D).
  • Islet1 and Pou4f2 are upstream of an RGC fate–inducing regulatory network, we assayed whether this combination of factors was able to induce multiple RGC genes in this cluster of MG-derived neurons.
  • We used the label transfer feature of Seurat to broadly compare the transcriptome of the IPA neurons to a reference dataset of all major retinal neuron classes (21, 22).
  • IPA-induced neurons a substantial number of RGC-associated genes were expressed in IPA-induced neurons (Fig.3D). These include genes such as Sox4 and Sox11, which are redundantly required for RGC fate acquisition (23, 24), and the axon growth–associated gene Gap43, which is highly expressed in developing RGCs (25).
  • Satb1 and Cntn5 were expressed in subsets of IPA-induced neurons. Satb1 is highly expressed in the ON- OFF direction-selective subtype of RGCs where it controls Cntn5 expression (26).
  • mice containing the tetO-Pou4f2-Islet1 and tetO-Ascl1-GFP cassettes to a germline Rosa26- rtTA line and then performed in vitro MG-reprogramming experiments (Fig.4, A and B).
  • MG were cultured from postnatal day 1111 mice for 7 days before passaging as previously described (10).
  • the cultures obtained in this way are largely composed of MG, although some surviving neurons are observed (10).5-Ethynyl-2′-deoxyuridine (EdU) was added to the medium to determine which cells are derived from proliferating MG and which cells were likely surviving neurons from the initial dissociation. After passage, doxycycline was added to the medium to induce transgene expression, and then cells were assayed with immunofluorescence and scRNA-seq (Fig.4B). [0184] Analysis of the cultures after 5 days of treatment confirmed that the cells express the transgenes. Immunolabeling for the transgenes showed that cells coexpress Pou4f2, Islet1, and Ascl1 (fig.12A).
  • the untreated MG were largely homogeneous, with one glial cluster and a small cluster containing only a few surviving neurons (fig.12, B and C).
  • Fig.4, F to I, and fig.12D we integrated all three together.
  • Fig.4, F to I, and fig.12D We identified cells in both the Ascl1-only in vitro and IPA- reprogrammed MG in vitro that mapped to the neuron clusters from the IPA in vivo dataset.
  • RGC genes such as Grin2a and Calb1 were found only in the IPA neurons (Fig.4J).
  • Fig.4J To identify unique marker genes expressed in the IPA neurons, we made a subset of all neuron populations in the IPA and Ascl1-only integrated dataset (Fig.4K) and performed differential gene expression (DGE) analysis (Fig. 4L).
  • DGE differential gene expression
  • Fig. 4L We identified a number of RGC genes enriched in the IPA neurons, while bipolar genes were enriched in the Ascl1 neurons.
  • canonical markers of the RGC lineage that were not induced in vivo, such as Sncg, Nefm, and Pou4f1 that were induced in vitro (Fig.4M).
  • IPA intrinsic photosensitive RGC marker
  • Ascl1 can stimulate MG-derived neurons that have physiological characteristics of endogenous retinal neurons, particularly bipolar cells (4, 13). Because IPA treatment leads to a different molecular and morphological neuronal phenotype compared to Ascl1 alone, we characterized the light responses and electrical properties of these cells.
  • FIG.5A glial-like hyperpolarized membrane potentials
  • Figure 5B shows examples of responses to families of current or voltage steps recorded from IPA-treated GFP+ cells. These cells exhibit a range of characteristics. Some have a neuronal phenotype and appear to express voltage-activated K+ conductance that limit the extent of depolarization to current steps, while others retain features of glia (Fig. 5B).
  • IPA increases the diversity of the electrical properties of the MG-derived neurons, including generating some cells that can produce Na+ and/or Ca2+ action potentials.
  • IPA expression remodels MG chromatin to an imperfect RGC-like fate
  • scATAC-seq transposase-accessible chromatin sequencing
  • Mice were treated with the same in vivo retinal regeneration paradigm described in Fig.3 but were processed for scATAC-seq instead of scRNA-seq.
  • Nuclei (1692) from Ascl1 only and 2451 nuclei from IPA treatment passed our quality control metrics (see Materials and Methods).
  • Fig.6, A to C Single cells from these two treatments were then integrated and plotted as a UMAP to identify cell types (Fig.6, A to C).
  • Cell type clusters were identified by the pattern of accessible chromatin near genes identified with specific retinal cell types. Coverage plots show representative peaks for the groups we identified: MG (Rlbp1+), neurogenic transition (Islet1+), MG-derived bipolar cells (Crx+), induced RGC-like cells (Pou4f2+), and photoreceptors (Arr3+) (Fig.6D).
  • the progenitor/MG marker Sox2 is still detectable by immunofluorescence 3 weeks after IPA reprogramming in GFP+ cells with neuronal morphology (Fig.6K).
  • Atoh1 can improve the ability of IPA to induce RGC-like cells from MG
  • the scRNA and scATAC-seq analysis revealed that, although IPA-induced neurons resembled RGCs, these cells lack some features of mature RGCs.
  • One hypothesis for why this is the case is the persistence of glial and progenitor genes and chromatin accessibility (i.e., Sox, Rax, Vsx2, etc.) in the MG-derived neurons.
  • Nonmammalian adult vertebrates can regenerate neurons in many regions of their central nervous system (CNS). For example, after tail amputation in larval frogs and some adult urodeles, the radial glial cells of the spinal cord acquire a pattern of gene expression similar to neuronal precursors and go on to proliferate and regenerate an apparently normal spinal cord (30). Similarly, in the retina and brain of zebrafish, glia respond to injury by activating a progenitor-like gene expression program of TFs (31). These glia-derived progenitor cells undergo multiple rounds of mitotic cell divisions, and the progeny differentiate into the range of neuron types that can restore function in the brain and retina (32).
  • CNS central nervous system
  • Reexpressing developmentally active TFs in adult mammalian glia can trigger a regenerative process in these cells that, in many ways, resembles what is found in fish and amphibians.
  • the transgenic overexpression of the proneural TF Ascl1 combined with histone deacetylase inhibition, can stimulate MG to acquire a progenitor-like state with the capacity of generating bipolar neurons (4).
  • the MG-derived neurons differentiate to the point that they make synapses with the surrounding neuronal circuitry and respond to light.
  • Ascl1 induces MG to adopt many features of retinal progenitors, including proliferative neurogenesis and a transcriptional and epigenetic landscape similar to developmental progenitors, not all developmentally appropriate Ascl1 targets are induced in MG-derived progenitor cells, and the neuronal output from Ascl1 MG is restricted to primarily bipolar neurons (5). Thus, we reasoned that additional TFs might be required to properly steer MG-derived progenitors to specific types of neurons. This is particularly important for endogenous regeneration strategies because most blinding diseases are the result of deficits in a particular neuronal subtype. For example, glaucoma is primarily caused by the death of RGCs.
  • RGCs are generated during development by a cascade of TFs, characterized by the initial expression of Atoh7 and the downstream expression of additional TFs, such as Pou4f1/2 and Islet1 (33). Atoh7 is necessary for proper RGC fate by inducing these downstream stabilizing TFs (34–36). Two of these downstream TFs, Pou4f2 and Islet1, are required for proper RGC fate specification (18, 37, 38), and ectopic expression of Pou4f2 and Islet1 in the Atoh7 null retina is sufficient to rescue the RGC fate (19).
  • MG-derived RGC-like cells express many RGC genes, they may lack some critical migration program. Nevertheless, some of these cells connect with the existing neural circuitry and respond to light, and it may be that appropriate connectivity can be established without proper somal location.
  • we also did not observe robust axonal outgrowth directed to the optic nerve we find that many genes important for axon growth and guidance are expressed in the RGC- like cells. It is possible that some key guidance factors are not expressed in these cells or, alternatively, that the adult retinal environment no longer expresses the guidance factors needed to direct axons to the optic nerve head.
  • FIGS.17A-17C illustrate the protocol used to demonstrate that the lineage of HuC/D+ neurons was traced from MG by tdTomato. Immunofluorescent markers showed successful delivery of reprogramming transcription factors resulting in HuC/D+ neurons reprogrammed from MG cells.
  • Atoh1 and Atoh7 delivered by AAV can induce HuC/D+ neurons. Atoh1 and Atoh7 are about the same in efficiency. AAV delivery of reprogramming factors under these conditions was much less efficient than transgenic expression.
  • Irx2 and Neurod2 promote axon growth in IPA reprogrammed MG (FIG.19). This shows that certain RGC transcription factors can increase some specific RGC genes.
  • Example 4 Human Muller glia can be generated in vitro from fetal retina or pluripotent stem cells
  • ESC Embryonic Stem cell
  • RO retinal organoids
  • FIG.20A Images of MG development in retinal organoids over time labeled with RLBP1 and SOX2 are shown in Fig.20B.
  • the upper panel of Fig.20C shows the schematic protocol for the generation of retinospheres (RS), and the lower panel shows images of retinospheres made from several fetal retinas and cultured for various times as labeled.
  • RS retinospheres
  • Fig.20D Characterization of the MG in RS with RLBP1, VSX2, SOX9, SOX2 and GFAP is shown in Fig.20D.
  • Human MG can be reprogrammed with ASCL1 to generate neurons in dissociated cultures (FIG.22). These neurons express some pan neuronal markers such as DCX and TUJ1, but are not mature.
  • Human MG can be derived from either retinal organoids or fetal human retina; both glial sources respond similarly to ASCL1.
  • Example 5 Promoters for driving expression in Muller glia
  • the characterization of ShH10 capsid and RLBP1 promoter in non-human primate (NHP) Muller glia is illustrated in FIG.23.
  • HES1 promoter provides a good alternative to RLBP1 promoter. HES1 promoter is only 337 bp, it is expressed in adult Muller glia, and in retinal progenitors. In addition, HES1 is increased in expression after ASCL1 infection. As shown in FIG.25, HES1 promoter drives very good expression specifically in Muller glia in human retinal organoids.
  • GFP is expressed in HES1 and Sox9 positive cells seven days after the infection with the Hes1-GFP construct. The construct is specific at 7 days.
  • Figure 25C shows that GFP is not expressed in OTX2 positive cells (HES1-GF), however we do obtain GFP/ OTX2 positive cells after the expression of ASCL1 (reprogrammed cells; Fig.25D).
  • HES1-promoter also directs GFP expression in MG in adult NHP dissociated cultures, as shown in FIG.26. [0271] This small and specific promoter offers the potential for a single AAV virus to drive reprogramming TFs in Muller glia.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Environmental Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des compositions, des molécules d'acide nucléique et des méthodes permettant d'induire une régénération rétinienne et une reprogrammation de la glie de Müller (MG) en cellules ganglionnaires rétiniennes chez un sujet. Des facteurs de transcription des cellules ganglionnaires rétiniennes (RGC) de croissance Pou4f2 et Islet1 augmentent la capacité neurogène induite par Ascl1 de la MG. L'association d'Ascl1, de Pou4f2 et d'Islet1 stimule la MG pour générer des cellules bipolaires et des neurones de type RGC. De même, le facteur de transcription Onecut1, qui est exprimé dans des cellules rétiniennes en développement, mais pas dans la MG, amène la MG à générer des cellules de type RCG. Des facteurs de transcription supplémentaires qui peuvent être utilisés comprennent Irx2, Irx5, Neurod2, Ebf1 et Tcf3. Les RGC dérivées de la MG peuvent présenter des potentiels d'action in vivo, et afficher des profils de chromatine similaires à des RGC de croissance.
PCT/US2023/065219 2022-04-01 2023-03-31 Méthode de stimulation de la régénération de cellules ganglionnaires rétiniennes WO2023192994A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263362361P 2022-04-01 2022-04-01
US63/362,361 2022-04-01

Publications (2)

Publication Number Publication Date
WO2023192994A2 true WO2023192994A2 (fr) 2023-10-05
WO2023192994A3 WO2023192994A3 (fr) 2024-02-29

Family

ID=88203588

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/065219 WO2023192994A2 (fr) 2022-04-01 2023-03-31 Méthode de stimulation de la régénération de cellules ganglionnaires rétiniennes

Country Status (1)

Country Link
WO (1) WO2023192994A2 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113134076A (zh) * 2020-01-16 2021-07-20 上海科技大学 一种利用转录因子再生具备功能的视网膜神经节细胞的方法

Also Published As

Publication number Publication date
WO2023192994A3 (fr) 2024-02-29

Similar Documents

Publication Publication Date Title
Bray et al. Thrombospondin-1 mediates axon regeneration in retinal ganglion cells
RU2707540C2 (ru) Композиции и способы для лечения гемоглобинопатий
White et al. A molecular toolbox for rapid generation of viral vectors to up-or down-regulate neuronal gene expression in vivo
WO2021031810A1 (fr) Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie du système nerveux liée à la mort neuronale fonctionnelle
CA2849720A1 (fr) Procedes de therapie genique ameliores
WO2021032068A1 (fr) Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie du système nerveux liée à la mort neuronale fonctionnelle
WO2012094193A2 (fr) Procédés pour améliorer l'administration de cellules transduites avec un gène
US8278284B2 (en) Therapeutic agents for diseases associated with apoptotic degeneration in ocular tissue cells that use SIV-PEDF vectors
CN112386699A (zh) Ptbp1抑制剂在预防和/或治疗功能性神经元死亡相关的神经***疾病中的应用
WO2023104028A1 (fr) Traitement de maladie neurologique médiée par un arn non codant
US20070041947A1 (en) Use of a lentiviral vector in the treatment of pain
US20220193265A1 (en) Methods and compositions for reprogramming müller glia
WO2023192994A2 (fr) Méthode de stimulation de la régénération de cellules ganglionnaires rétiniennes
US20210228741A1 (en) Methods and compositions to stimulate retinal regeneration
US20220098255A1 (en) Neurod1 combination vector
CA3132420A1 (fr) Constructions d'expression pour la modification genetique de cellules
US20220098616A1 (en) ISL1 and LHX3 VECTOR
US20220098617A1 (en) Ascl1 vector
US20220098254A1 (en) NEUROD1 and DLX2 VECTOR
WO2023150553A1 (fr) Ciblage et transduction basés sur un promoteur rcpg17 de cellules progénitrices gliales
Stieglitz The control of neuronal activity and behavior by members of the HCN and TPC family
KR20230123460A (ko) Neurod1 벡터
CN116761812A (zh) Neurod1和dlx2载体
CA3211054A1 (fr) Therapie genique pour le syndrome de deficience en transporteur de la dopamine
CA3157523A1 (fr) Regeneration de neurones fonctionnels pour le traitement d'une lesion de la moelle epiniere et d'une sla

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23782100

Country of ref document: EP

Kind code of ref document: A2